Procedures have been developed for the use of vaccinia virus as a eukaryotic expression vector. A chimeric gene is formed by ligating vaccinia virus transcriptional regulatory signals to a foreign protein coding sequence. Homologous recombination is used to insert the chimeric gene into a non-essential region of the vaccinia virus genome. To facilitate the formation and isolation of recombinant virus, new plasmid vectors have been constructed which direct the insertion of the chimeric gene together with the E. coli Beta-galactosidase gene into the thymidine kinase locus. Recombinant virus is then selected on the basis of the thymidine kinase negative phenotype and/or staining with a Beta-galactosidase indicator dye. The recombinant viruses produced in this manner are stable and have a wide host range for tissue culture cells and animals. At least 25,000 base pairs of DNA can be inserted into the vaccinia virus genome without destroying infectivity. To optimize expression, the prokaryotic gene encoding chloramphenicol acetyltransferase was inserted into vaccinia virus under control of different promoters. This system has been used to express genes from a variety of infectious agents including herpes simplex virus type 1, hepatitis B virus, influenza virus, vesicular stomatitis virus and rabies virus. Animals vaccinated with each of the above recombinants were protected against challenge with the corresponding virus.